Grading, staging, and prognosing cancer using osteopontin-c

ABSTRACT

The present disclosure provides methods and kits that can be used to determine the grade or stage of a breast or other cancer, such as a ductal carcinoma in situ (DCIS). By determining the grade, stage, or aggressiveness of a cancer, appropriate therapeutic regiments can be selected and administered to the patient with the cancer. The method includes detecting osteopontin-c (OPN-c), wherein the presence of high amounts of OPN-c in the cancer sample indicates that the subject has a more aggressive form of cancer (e.g., grade 3).

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 12/682,310,now U.S. Pat. No. 9,873,915, which is a National Stage Entry ofPCT/US08/80162, filed Oct. 16, 2008, which claims priority to U.S.Provisional Application No. 60/980,379 filed Oct. 16, 2007, each ofwhich is herein incorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This study was supported by Department of Defense breast cancer grantDAMD17-02-0510 and USPHS grant M01 RR 08084 from the General ClinicalResearch Centers Program, National Center for Research Resources, NIH.Therefore, the United States Government has certain rights to thisinvention.

FIELD

This application relates to methods for grading, staging, andprognosticating cancers, such as breast cancer, for example ductalcarcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS), and kitsthat can be used for such methods.

BACKGROUND

Around 20% of women who undergo breast biopsy procedures are diagnosedwith breast cancer on the basis of conventional histological evaluation.Most biopsies are obtained with a core needle, and sampling orinterpretation error may understate the disease identified. Furthermore,the early stages of breast cancer (ductal carcinoma in situ (DCIS) orlobular carcinoma in situ (LCIS)) are difficult to differentiate frombenign growths on one end of the spectrum and invasive cancers on theother end. There is a 30-50% risk that DCIS, if not treated, willprogress to breast carcinoma. Currently, there are no molecular markersat hand that identify this fraction of high risk DCIS cases. This makesit difficult to decide whether a patient should be treated withlumpectomy, lumpectomy plus radiation or hormone therapy, or resectionof the entire breast. While research has identified a large number ofbiomolecules to be deregulated or defective in breast cancer, relativelyfew of them are commonly used in histological diagnosis. Specifically,markers that predict invasiveness have not been firmly established.Better molecular predictors of progression are needed to facilitaterational treatment decisions.

Several markers are commonly used in breast cancer diagnosis. The U.S.Food and Drug Administration-approved and ASCO-recommended tumor markersCA15.3, CA27.29, and CEA are useful only for monitoring the therapy ofadvanced breast cancer or its recurrence. These serum markers still lackthe adequate sensitivity (below 25%) and specificity (below 70%) to beapplicable in detecting early stage breast carcinoma in a largepopulation [1, 2]. Estrogen receptor (ER) and progesterone receptor (PR)facilitate decisions on therapy, but are weak prognostic measures [2-4].HER2 over-expression is associated with poor prognosis, but thismechanism underlies only about one third of breast cancers.

Few tumor markers are under study as predictors of breast cancerprogression. The transcript and protein of the polycomb grouptranscriptional repressor EZH2 are consistently elevated in invasivebreast carcinoma compared with normal breast epithelia. The EZH2 proteinlevels are strongly associated with breast cancer aggressiveness [5].VEGF correlates strongly with uPA in the node-positive population.Patients with high VEGF levels display poor outcome, with an increasedrisk for the node-positive subset. Furthermore, infiltrating ductalcarcinomas express higher levels of both uPA and VEGF than intralobularcarcinomas [6]. Multiple malignant breast cancer cell lines over-expressthe metastasis gene osteopontin, and transfection of this gene intobenign tumorigenic breast epithelial cell lines conveys invasivebehavior [7]. Consequently, osteopontin has been identified as aprognostic marker associated with patient survival. However, osteopontinmay also be present in non-tumorous tissue and in the plasma of healthyindividuals [8, 9].

Osteopontin may serve as a cytokine and as an extracellular matrixmolecule. It can support migration and protect from programmed celldeath after ligation of certain integrin receptors or a CD44 variant onthe cell surface. The biological functions of metastasis-associated geneproducts are extensively regulated on the post-transcriptional andpost-translational levels [10]. Consistently, osteopontin secreted fromvarious cells has diverse structural characteristics [11, 12] andtumor-derived osteopontin forms are smaller than osteopontin secreted bynon-transformed cells [13]. Transcripts for three osteopontin splicevariants are expressed in invasive, but not in non-invasive, breasttumor cell lines [14].

Alternative splicing of osteopontin occurs upstream of the centralintegrin binding domain and the C-terminal CD44 binding domain.Osteopontin-b lacks exon 5 and osteopontin-c lacks exon 4.

SUMMARY

While the acquisition of invasiveness is a critical step in early stagebreast carcinomas (such as CIS), no established molecular markersreliably identify tumor progression. The metastasis gene osteopontin(OPN) is subject to alternative splicing, which yields three messages,osteopontin-a (OPN-a), osteopontin-b (OPN-b), and osteopontin-c (OPN-c).It is shown herein that OPN-c is a biomarker for breast cancer. The RNAmessage for OPN-c was present in 16 of 20 breast cancers (80%), but wasundetectable in 22 normal specimens obtained from reduction mammoplasty.In contrast, OPN-a RNA was expressed at various levels in all 20 breastcancers, eleven tumor-surrounding tissues, and 21 normal samples. Thesplice variant OPN-b was present at barely detectable levels in 18 of 20cancers and in six of 22 normal breasts. By immunohistochemistry, 66 of69 normal breasts were negative, while three showed low level stainingAmong the breast cancers, 43 of 56 cores (77%) stained positive forOPN-c. When correlated with tumor grade, the staining for OPN-cincreased from grade 1 to grade 3. OPN-c detects a higher fraction ofbreast cancers than estrogen receptor (ER), progesterone receptor (PR),or human epidermal growth factor receptor 2 (HER2). In 178 breastspecimens analyzed, OPN-c was present in 78% of cancers, 36% ofsurrounding tissues, and 0% of normal tissues. Therefore, OPN-c canserve as a selective diagnostic and prognostic marker for breast cancer.It can also be used in a diagnostic panel together with conventionalbreast cancer markers such as ER, PR and HER2.

Based on these observations, methods for grading, staging, and orprognosticating breast cancer (such as CIS), or other tumor thatexpresses osteopontin (such as OPN-c), in a subject are provided. Inparticular examples the method includes detecting or measuring OPN-c ina breast or other cancer sample, wherein the presence of relatively highOPN-c levels indicates that the subject has a higher grade (e.g., a moreaggressive form of) breast or other cancer, such as a grade 3 breastcancer. In contrast, if relatively low OPN-c levels are detected in thebreast or other cancer sample, this indicates that the subject has alower grade (e.g., a less aggressive form of) breast or other cancer,such as grade 1. In some examples, when referring to tumor grades herein(i.e., grade scale of 1-3) the tumor grading is as per theScarff-Bloom-Richardson system [15, herein incorporated by reference asto the grading method and descriptions]. However, one skilled in the artwill appreciate that other methods can be used, and that variations inscoring may occur between samples or when different detection reagentsare used. In some examples, the greater the amount of OPN-c detected(e.g., 3 on a scale of 0 to 3) indicates that the subject has a highergrade (or more aggressive form of) breast or other cancer, such as agrade 3 cancer, while the lower the amount of OPN-c detected (e.g., 1 ona scale of 0 to 3) indicates that the subject has a lower grade (or lessaggressive form of) breast or other cancer, such as grade 1. Any methodof detecting OPN-c can be used, such as methods that permit detection ofOPN-c proteins (e.g., by using OPN-c antibodies) or OPN-c nucleic acidmolecules (e.g., by using OPN-c nucleic acid probes or primers).

The method can further include detecting or measuring one or more othercancer markers in the sample, such as the breast cancer markers ER; PR;and/or HER2. For example, low levels of ER or high levels of HER2 andsignificantly elevated OPN-c (an in some examples also low PR) in thebreast cancer sample indicates that the subject a grade 2 or 3 breastcancer while lower levels of OPN-c and in some examples also lowerlevels of HER2 (and in some examples if the sample is also ER+ and/orPR+) in the breast cancer sample indicates that the subject has a grade1 breast cancer.

Based on the results of the method, a treatment protocol can be selectedfor the subject. Currently, there is no particular treatment regimen fora subject who is shown to be OPN-c positive. However, based on the dataprovided herein, subjects shown to have a more aggressive form of breastor other cancer (for example even if they are negative for ER, PR, orHER2) can receive a more aggressive therapy (such as one or more ofmastectomy, chemotherapy, radiation) than subjects shown to have a lessaggressive form of the cancer.

In particular examples, the method has a sensitivity of at least 80% anda specificity of at least 80%, such as a sensitivity of at least 90% anda specificity of at least 90%.

The present disclosure also provides kits that can be used for breast orother cancer diagnosis, staging, grading, or combinations thereof. Forexample, such kits can be used to identify a subject having aggressiveor less aggressive forms of breast cancer. In particular examples, suchkits include or consist of specific binding agents (such as antibodies)that bind to one or more of ER, PR, HER2, and OPN-c. In some examples,the kits include or consist of nucleic acid primers or probes specificfor OPN-c, ER, PR, and HER2. The kits can also include one or morecontrol samples (such as positive and negative control samples) whosedetectable levels of ER, PR, HER2 and/or OPN-c and/or grade of breastcancer are known.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C: OPN splice variant expression in breast tissue. A) RNA wasanalyzed by real-time RT-PCR from snap frozen tissue samples. The dataare presented as mean±SEM. The total number of samples and the number ofsamples with undetectable levels of osteopontin over 40 cycles ofamplification is indicated above the graph. B) Representative agarosegel with the real-time RT-PCR products of a tumor specimen. C)Reproducibility of real time RT-PCR amplification. MDA-MB-435 cells,known to express OPN-a and the splice variants -b and -c [14], were usedas control for calculating the expression of osteopontin forms in thetissue. β-actin served as a reference gene and relative expressionratios of the target gene were calculated from the cycle threshold andefficiency measurements (Pfaffl, Nucleic Acids Res 29:e45, 2001).Repeated thawing of cDNA degrades the messages for osteopontin splicevariants faster than the message for β-actin. The breast specimens wereobtained and analyzed over two time periods interrupted by a break ofseveral months. The scatters of the reference values within each timeperiod were tighter than the combined ranges, possibly reflecting asmall influence by batch-to-batch variations in the reagents used forRNA extraction, reverse transcription, and amplification.

FIGS. 2A-C: Antibody characterization. A) Specificity of anti-hOPNc IgY.Hens were immunized with a peptide representing the splice junction ofhuman OPN-c. To mimic an internal sequence, the peptide was N-terminallyacetylated. At the end of the immunization period, total immunoglobulin(IgY) was purified from eggs and used for Western blotting. Left panel:supernatants from transfected MCF-7 cells probed with the anti-hOPNcantibody (a prior Western blot of the same supernatants had demonstratedthe comparable expression levels of OPN-a and -c). Right panel: 400 ngof GST-OPN was loaded per lane, probed with the anti-hOPNc antibody. Theadditional band around 120 kD is commonly observed for osteopontin andlikely reflects an aggregated form of the molecule. The purity of theGST-OPN fusion proteins is shown on the Coommassie gel. Osteopontin-aand -c were amplified by PCR by exclusion of the signal peptide (aminoacids 1-17). The amplified fragments were subcloned into pGEX-5T vectorand transformed into BL21 bacteria for protein synthesis. Reading framesand sequence fidelity were confirmed by sequencing analysis. Theproteins were purified from bacterial lysates by pull-down withGSH-Sepharose. B) Immunocytochemistry of cell lines with known OPNexpression levels. MCF-7 cells and ZR-75 cells are non-invasive and donot express osteopontin, MDA-MB-435 cells are metastatic in vivo andexpress all three osteopontin splice variants [14]. C) Staining ofbreast cancer tissue with the IgY antibody to osteopontin-c (top left)or with a rabbit antibody to a common region of all splice variants(0-17, Assay Designs Inc.) (bottom left). The negative controls (top andbottom right) reflect the work-up without the primary antibody.

FIGS. 3A-C: Expression of osteopontin-c protein in breast tissue. Tissuearrays were stained by immunohistochemistry and scored by aboard-certified pathologist. A) Combined pathology scores (meanintensity multiplied by mean percent of tissue stained) for theindividual samples of the normal breast array (top) and the breastcarcinoma array (bottom). Note that the highest positively scoring core,number 7, in the normal breast array was judged to be possiblycancerous. B) Representative images (taken at 40× magnification) of theimmunohistochemical staining for OPN-c. Increasing grades are shown forintraductal carcinoma, infiltrating lobular carcinoma, and papillaryadenocarcinoma. The insert depicts a normal breast tissue specimen as areference. The slides are counterstained with Hematoxylin II and BluingReagent (Ventana Medical Systems Inc.). C) Pathology scores for tumorsof increasing grade (left panel) or of tumors versus normal breasts(right panel). Shown are the mean cytoplasmic combined score (top row),the mean percent positive cells (middle row), and the mean cytoplasmicintensity (bottom row). The error bars represent SEM.

FIGS. 4A-C: OPN-c is a more sensitive marker for breast cancer than ER,PR, or HER2. A) Pathology scores for tumors of increasing grade. Shownare the mean combined scores for ER (top), PR (middle) and HER2(bottom). The error bars represent SEM. B) Spearman's rank correlationcoefficients (SRCC) and associated confidence parameters for thecorrelation of breast carcinoma staining for OPN-c with the stainingobserved for ER, PR and HER2 by immunohistochemistry. The scatter plotsshow the combined mean scores for osteopontin-c versus ER, PR or HER2.C) Threshold probabilities for the best performing logistic regressionmodel for predicting breast cancer grades 2 or 3 with a combination ofOPN-c, ER, and HER2.

SEQUENCE LISTING

Applicant hereby incorporates by reference a CRF sequence listingsubmitted herewith having a file name 25033us2_ST25 created on Jan. 9,2018 and a size of 3,866 bytes.

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NOS: 1 and 2 are exemplary forward and reverse primers,respectively, that can be used to amplify OPN-a.

SEQ ID NOS: 3 and 4 are exemplary forward and reverse primers,respectively, that can be used to amplify OPN-b.

SEQ ID NOS: 5 and 6 are exemplary forward and reverse primers,respectively, that can be used to amplify OPN-c.

SEQ ID NOS: 7 and 8 are exemplary forward and reverse primers,respectively, that can be used to amplify β-actin.

SEQ ID NOS: 9 and 10 are exemplary forward and reverse primers,respectively, that can be used to amplify Ck-19.

SEQ ID NOS: 11 and 12 are exemplary forward and reverse primers,respectively, that can be used to amplify aP2.

SEQ ID NOS: 13 and 14 are exemplary forward and reverse primers,respectively, that can be used to amplify GAPDH.

SEQ ID NOS: 15 and 16 are exemplary forward and reverse primers,respectively, that can be used to amplify the osteopontin codingsequence.

DETAILED DESCRIPTION Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising anantibody” includes single or plural antibodies and is consideredequivalent to the phrase “comprising at least one antibody.” The term“or” refers to a single element of stated alternative elements or acombination of two or more elements, unless the context clearlyindicates otherwise. As used herein, “comprises” means “includes.” Thus,“comprising A or B,” means “including A, B, or A and B,” withoutexcluding additional elements. Dates of GenBank Accession Nos. referredto herein are the sequences available at least as early as Oct. 15,2007, and are hereby incorporated by reference.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

CIS: carcinoma in situ

DCIS: ductal carcinoma in situ

ER: estrogen receptor

HER2: human epidermal growth factor receptor 2

LCIS: lobular carcinoma in situ

OPN: osteopontin

PR: progesterone receptor

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, that is, molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen(such as OPN-c, ER, PR, or HER2). Exemplary antibodies includemonoclonal, polyclonal, and humanized antibodies.

A naturally occurring antibody (such as IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. As used herein, the term antibodyalso includes recombinant antibodies produced by expression of a nucleicacid that encodes one or more antibody chains in a cell (for example seeU.S. Pat. Nos. 4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison,J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239,1984).

The term antibody also includes an antigen binding fragment of anaturally occurring or recombinant antibody. Specific, non-limitingexamples of binding fragments encompassed within the term antibodyinclude Fab, (Fab′)₂, Fv, and single-chain Fv (scFv). Fab is thefragment that contains a monovalent antigen-binding fragment of anantibody molecule produced by digestion of whole antibody with theenzyme papain to yield an intact light chain and a portion of one heavychain or equivalently by genetic engineering. Fab′ is the fragment of anantibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule.(Fab′)₂ is the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction orequivalently by genetic engineering. F(Ab′)₂ is a dimer of two FAb′fragments held together by disulfide bonds. Fv is a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains.Single chain antibody (“SCA”) is a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Methods of making thesefragments are routine in the art.

Binding affinity: Affinity of an antibody for an antigen, such as theaffinity of an antibody for an OPN-c peptide. In one example, affinityis calculated by a modification of the Scatchard method described byFrankel et al., Mol. Immunol., 16:101-106, 1979. In another example,binding affinity is measured by an antigen/antibody dissociation rate.In yet another example, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity is at least about 1×10⁻⁸ M. In other example, a high bindingaffinity is at least about 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at leastabout 2.5×10⁻⁸, at least about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, atleast about 4.0×10⁻⁸, at least about 4.5×10⁻⁸, or at least about5.0×10⁻⁸ M.

Breast cancer: Includes any tumor of the breast, such as tumors ofepithelial (carcinoma) or stromal (sarcoma) breast tissue. Exemplary insitu epithelial breast cancers include DCIS and LCIS. Lobular carcinomain situ (LCIS) is a tumor that consists of abnormal cells in the liningof a lobule. Subjects having LCIS means that the subject has anincreased risk of developing breast cancer in either breast. Ductalcarcinoma in situ (DCIS) is made up of abnormal cells in the lining of aduct. It is a pre-invasive malignant tumor, and is also calledintraductal carcinoma. The abnormal cells have not yet spread beyond theduct and have not yet invaded the surrounding breast tissue. DCIS issometimes called Stage 0 breast cancer because it is not invasive.Exemplary invasive breast carcinomas include carcinoma NOS (nototherwise specified), lobular carcinoma, tubular/cribriform carcinoma,mucinous (colloid) carcinoma, medullary carcinoma, papillary carcinoma,and metaplastic carcinoma. An exemplary breast sarcoma is phyllodestumour.

Contact: To bring one agent into close proximity to another agent,thereby permitting the agents to interact. For example, an antibody canbe applied to a microscope slide or other surface containing abiological sample, thereby permitting detection of one or more proteinsin the sample that are specific for the antibody.

Detect: To determine if an agent is present or absent. In some examplesthis can further include quantification. For example, use of an antibodyspecific for a particular protein (e.g., OPN-c, ER, PR, or HER2) permitsdetection of the protein in a sample, such as a sample containing breastcancer tissue. In particular examples, an emission signal from afluorophore (such as an increase in the signal) is detected.

Detection can be in bulk, so that a macroscopic number of molecules(such as at least 10²³ molecules) can be observed simultaneously.Detection can also include identification of signals from singlemolecules using microscopy and such techniques as total internalreflection to reduce background noise.

Estrogen receptor (ER): A member of the nuclear hormone family ofintracellular receptors is activated by 17β-estradiol. Estrogenreceptors, such as ERα, are overexpressed in around 70% of breast cancercases, referred to as “ER positive” (ER+). There are two different formsof the estrogen receptor, usually referred to as α and β, each encodedby a separate gene (ESR1 and ESR2 respectively). ERα (OMIM 133430),expressed from the ESR1 gene, is found in endometrium, breast cancercells, ovarian stroma cells and in the hypothalamus. Therefore, inparticular examples ER refers to the ERα form found in breast cancercells.

Estrogen receptor sequences are publicly available. For example, GenBankAccession Nos: BC128574.1 (nucleic acid) and P03372.2 (protein) disclosehuman ERα sequences and Nos. NM_007956.4 (nucleic acid) and P19785.1(protein) disclose mouse ERα sequences. In one example, an ER sequenceincludes a full-length wild-type (or native) sequence, as well asvariants (e.g., polymorphisms) found in breast cancer cells. In certainexamples, ER has at least 80% sequence identity, for example at least85%, at least 90%, at least 95%, or at least 98% sequence identity to anative ERα (such as the sequence of GenBank Accession Nos: P03372.2,BC128574.1, NM_007956.4 and P19785.1) and retains ERα activity (e.g.,expressed in breast cancer cells). In other examples, an ER nucleic acidsequence has a sequence that hybridizes under very high stringencyconditions to a sequence set forth in GenBank Accession No. BC128574.1or NM_007956.4 and is expressed in breast cancer cells.

Grading cancer: A cancer can be graded according to its level ofdifferentiation. The lower the number, the lower the grade, and theslower the cancer is growing. Cancers are usually graded on a scale of 1to 3. Grade 1 indicates the cancer cells look similar to normal cells,and the cancer is likely to be less aggressive. Grade 2 indicates thecancer cells appear dysplastic, and are more likely to be aggressive andgrow faster. Grade 3 cancer cells highly dysplastic and are more likelyto be very aggressive in growth.

Human epidermal growth factor receptor 2 (HER2): A member of the ErbBprotein family, which is a proto-oncogene. In humans, the HER2 gene islocated at the long arm of human chromosome 17(17q11.2-q12).Approximately 25-30% of breast cancers have an amplification of theHER2/neu gene or overexpression of its protein product, referred to as“HER2 positive” (HER2+). HER2+ patients can receive the monoclonalantibody trastuzumab (Herceptin) as a therapy for breast cancer.Overexpression of HER2 in breast cancer has been associated withincreased disease recurrence and worse prognosis.

HER2 sequences are publicly available. For example, GenBank AccessionNos: NM_001005862.1 and NM_004448.2 (nucleic acid) and NP_001005862.1and NP_004439.2 (protein) disclose human HER2 sequences. In one example,a HER2 sequence includes a full-length wild-type (or native) sequence,as well as HER2 variants (e.g., polymorphisms) found in breast cancercells. In certain examples, HER2 has at least 80% sequence identity, forexample at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to a native HER2 (such as the sequence of GenBankAccession Nos: NM_001005862.1, NM_004448.2, NP_001005862.1 andNP_004439.2) and retains HER2 activity. In other examples, a HER2nucleic acid sequence has a sequence that hybridizes under very highstringency conditions to a sequence set forth in GenBank Accession No.NM_001005862.1 or NM_004448.2 and retains HER2 activity.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, one or more labels can beattached to an antibody, thereby permitting detection of the targetprotein. In another example, one or more labels can be attached to anucleic acid probe, thereby permitting detection of the target nucleicacid molecule. Exemplary labels include radioactive isotopes,fluorophores, ligands, chemiluminescent agents, enzymes, andcombinations thereof.

Normal cells or tissue: Non-tumor, non-malignant cells and tissue.

Nucleic acid molecules: A deoxyribonucleotide or ribonucleotide polymerincluding, without limitation, cDNA, mRNA, genomic DNA, and synthetic(such as chemically synthesized) DNA. The nucleic acid molecule can bedouble-stranded or single-stranded. Where single-stranded, the nucleicacid molecule can be the sense strand or the antisense strand. Inaddition, nucleic acid molecule can be circular or linear. Thedisclosure includes methods that detect OPN-c, ER, PR, and HER2 nucleicacid molecules.

Osteopontin-c (OPN-c): Osteopontin is a secreted multi-functionalphosphorylated glycoprotein expressed at high levels in tumors,including those of the breast. Several splice variants of OPN have beenidentified, including OPN-a (native sequence), OPN-b (truncatedsequence) and OPN-c (truncated sequence). OPN-c lacks exon 4 (27 aminoacids) in the NH₂-terminal region of the mature sequence. OPN-c lacksthe transglutaminase reactive domain (Gly-X-Gly) which can mediatecovalent homodimer cross-linking as well as heterodimer formation toother matrix components (such as fibronectin).

OPN-c sequences are publicly available. For example, GenBank AccessionNos: D28761 (nucleic acid) and BAA05951 (protein) disclose human OPN-csequences. In one example, an OPN-c sequence includes a wild-type (ornative) sequence, as well as OPN-c variants (e.g., polymorphisms)expressed in breast cancer cells. In certain examples, OPN-c has atleast 80% sequence identity, for example at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to a native OPN-c (such asthe sequence of GenBank Accession Nos: D28761 and BAA05951). In otherexamples, an OPN-c nucleic acid sequence has a sequence that hybridizesunder very high stringency conditions to a sequence set forth in GenBankAccession No. D28761 and retains OPN-c activity.

Primer: Short nucleic acid molecules, for instance DNA oligonucleotides10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50nucleotides or more in length, such as this number of contiguousnucleotides of a OPN-c, ER, PR, or HER2 nucleic acid molecule (e.g.,gene, cDNA, or mRNA sequence). Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand. Primer pairs can beused for amplification of a nucleic acid sequence, such as by PCR orother nucleic acid amplification methods known in the art.

Methods for preparing and using nucleic acid primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), and Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of ordinary skill in theart will appreciate that the specificity of a particular primerincreases with its length.

In one example, a primer includes at least 15 consecutive nucleotides ofan OPN-c, ER, PR, or HER2 nucleic acid molecule, such as at least 18consecutive nucleotides, at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50 or more consecutivenucleotides of an OPN-c, ER, PR, or HER2 nucleotide sequence. Suchprimers can be used to amplify OPN-c, ER, PR, or HER2, for example usingPCR.

Probe: A short sequence of nucleotides, such as at least 8, at least 10,at least 15, at least 20, at least 21, at least 25, or at least 30nucleotides in length (e.g., 8 to 40 or 10 to 30 nucleotides), used todetect the presence of a complementary sequence (such as a OPN-c, ER,PR, or HER2 nucleic acid sequence), for example by molecularhybridization. Ideally, a probe that is specific for a particularnucleic acid sequence (e.g., an OPN-c sequence) does not significantlyhybridize to other nucleic acid sequences (e.g., non-OPN-c sequences)under highly stringent conditions. In particular examples,oligonucleotide probes include a label that permits detection ofoligonucleotide probe:target sequence hybridization complexes. Forexample, an oligonucleotide probe can include these numbers ofcontiguous nucleotides of an OPN-c, ER, PR, or HER2 nucleic acidmolecule (e.g., gene, cDNA, or mRNA sequence), along with a detectablelabel. Such an oligonucleotide probe can be used to detect the presenceof the OPN-c, ER, PR, or HER2 nucleic acid molecule in a sample.

Progesterone receptor (PR): An intracellular steroid receptor thatspecifically binds progesterone. Progesterone receptors areoverexpressed in some breast cancer cases, referred to as “PR positive”(PR+).

The term progesterone receptor (OMIM: 607311) includes thoseprogesterone receptor genes (PGR) found in mammals and retainsprogesterone receptor biological activity. Progesterone receptorsequences are publicly available. For example, GenBank Accession Nos:AF016381.1 (nucleic acid) and AAD01587.1 (protein) disclose human PRsequences and Nos. M68915.1 (nucleic acid) and AAA39971.1 (protein)disclose mouse PR sequences. In one example, a PR sequence includes afull-length wild-type (or native) sequence, as well as PR variants(e.g., polymorphisms) that retain PR biological function (e.g., abilityto bind progesterone). In certain examples, PR has at least 80% sequenceidentity, for example at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to a native PR (such as the sequence ofGenBank Accession Nos: AF016381.1, AAD01587.1, M68915.1 and AAA39971.1)and retains PR activity. In other examples, a PR nucleic acid sequencehas a sequence that hybridizes under very high stringency conditions toa sequence set forth in GenBank Accession No. AF016381.1 or M68915.1 andretains PR activity.

Quantify: To express as a numerical amount, whether an actual amount ora relative amount.

Sample: A biological specimen containing genomic DNA, RNA (includingmRNA), protein, or combinations thereof, obtained from a subject.Examples include, but are not limited to, peripheral blood, urine,saliva, tissue biopsy, surgical specimen, and autopsy material. In someexamples, the sample is a tissue sample obtained from a subject known tohave, or suspected to have, cancer. In one example, a sample includesbreast tissue, such as that obtained during a needle biopsy, lumpectomy,or mastectomy. Samples, such as tissue samples, can be placed onmicroscope slides. In particular examples, samples are used directly, orcan be manipulated prior to use, for example, by fixing (e.g., usingformalin) or embedding (e.g., in plastic or paraffin).

Specific binding agent: An agent that binds substantially only to adefined target. Thus an OPN-c specific binding agent is an agent thatbinds substantially to an OPN-c peptide or nucleic acid molecule. In oneexample, the specific binding agent is an antibody that specificallybinds an OPN-c peptide.

The term “specifically binds” refers, with respect to an antigen such asOPN-c, to the preferential association of an antibody or other specificbinding agent, in whole or part, to the antigen and not to otherantigens. A certain degree of non-specific interaction can occur betweena specific binding agent and a non-target antigen. Nevertheless,specific binding can be distinguished as mediated through specificrecognition of the antigen. Specific binding results in a significantassociation between the antibody (or other specific binding agent) andthe antigen than between the antibody and a non-antigen. Specificbinding typically results in greater than 2-fold, such as greater than5-fold, greater than 10-fold, or greater than 100-fold increase inamount of bound antibody or other specific binding agent to the antigenas compared to binding to a non-specific antigen.

The determination that a particular agent binds substantially only toOPN-c or other protein (e.g., ER, PR, or HER2) can be made using oradapting routine procedures. For example, western blotting can be usedto determine that a specific binding agent, such as an antibody, bindssubstantially only to the protein (such as substantially only bindsOPN-c but not to other proteins in a breast cancer cell) (for examplesee Harlow and Lane, Antibodies: A Laboratory Manual. 1988). A varietyof immunoassay formats are appropriate for selecting antibodies or otherspecific binding agent specifically immunoreactive with a particularprotein (such as OPN-c). For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein.

Staging cancer: A cancer, such as breast cancer, can be staged todescribe the extent or severity of a cancer based on the extent of theoriginal (primary) tumor and the extent of spread in the body. Stagingconsiders the primary tumor (T), the regional lymph nodes (N), anddistant metastases (M). The size of the primary tumor and the extent ofregional lymph node metastases are usually staged on a scale from 0 to4. Metastases are staged as 0 (no metastases present) or 1 (metastasespresent). Low numbers indicate tumors that have spread little, whilehigh numbers refer to tumors that have spread extensively. The TNMclassification is most commonly used (see the AJCC Staging Manual),where T describes the size of the tumor and whether it has invadednearby tissue, N describes any lymph nodes that are involved, and Mdescribes metastasis (spread of cancer from one body part to another).However, other classification frameworks for staging cancer are withinthe scope of this disclosure.

In particular examples, the stages of breast cancer are as follows:Stage 0—carcinoma in situ; Stage I—Tumor (T) does not involve axillarylymph nodes (N); Stage IIA—T 2-5 cm, N negative, or T<2 cm and Npositive; Stage IIB—T>5 cm, N negative, or T 2-5 cm and N positive (<4axillary nodes); Stage IIIA—T>5 cm, N positive, or T 2-5 cm with 4 ormore axillary nodes; Stage IIIB—T has penetrated chest wall or skin, andmay have spread to <10 axillary N; Stage IIIC—T has >10 axillary N, 1 ormore supraclavicular or infraclavicular N, or internal mammary N; andStage IV—Distant metastasis (M).

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals, such as veterinary subjects. In aparticular example, a subject is one who had or is suspected of havinghad breast cancer, such as DCIS.

Target molecule: A biomolecule whose detection or measurement isdesired, such as a breast cancer marker. Examples of target moleculesinclude OPN-c, ER, PR, and HER-2.

Tumor: A neoplasm. In one example, a tumor is one that expresses OPN,such as OPN-c. Exemplary tumors that express OPN include but are notlimited to adrenocortical cancer, ameloblastoma, ampullary cancer,bladder cancer, bone cancer, breast cancer, cervical cancer,cholangioma, colorectal cancer, endometrial cancer, esophageal cancer,gastric cancer, glioma, granular call tumor, head and neck cancer,hepatocellular cancer, hydatiform mole, lung cancer, lymphoma, melanoma,mesothelioma, myeloma, neuroblastoma, oral cancer, osteochondroma,osteosarcoma, ovarian cancer, pancreatic cancer, pilomatricoma, prostatecancer, renal cell cancer, salivary gland tumor, soft tissue tumors,Spitz naevus, squamous cell cancer, teratoid cancer, and thyroid cancer.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity (e.g., appropriate time,temperature, reaction conditions). An example includes contacting anantibody or nucleic acid probe with a breast cancer sample sufficient toallow detection of one or more target molecules (e.g., ER, PR, HER2,OPN-c) in the sample and can include quantification of one or moretarget molecules in the sample.

Methods of Grading and Staging Cancer

It is shown herein that the OPN-c splice variant is expressed in 75-80%of breast carcinomas, but not in normal breast tissues. The levels ofOPN-c correlate with tumor grade. This makes OPN-c a marker for theinvasive potential of breast tumors, which can permit clinicians toselect appropriate treatment regimens for a patient with breast or othercancer. OPN-c is a better breast cancer marker than OPN-a because it isabsent from normal breast tissue. In fact, quantitative analysis of theRNA levels did not discern statistically significant differences betweennormal and tumorous breasts for OPN-a. Based on these results, detectionof OPN-c in a tumor sample can be used to grade or stage anyOPN-expressing tumor, such as adrenocortical cancer, ameloblastoma,ampullary cancer, bladder cancer, bone cancer, breast cancer, cervicalcancer, cholangioma, colorectal cancer, endometrial cancer, esophagealcancer, gastric cancer, glioma, granular call tumor, head and neckcancer, hepatocellular cancer, hydatiform mole, lung cancer, lymphoma,melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer,osteochondroma, osteosarcoma, ovarian cancer, pancreatic cancer,pilomatricoma, prostate cancer, renal cell cancer, salivary gland tumor,soft tissue tumors, Spitz naevus, squamous cell cancer, teratoid cancer,and thyroid cancer.

The lack of reliable molecular indicators for breast cancer progressionhas led to efforts to use increasingly complex readouts. Multiple tumormarkers, including HER2 amplification/over-expression, cathepsin D, anduPAR, have been considered for prognostication and therapy decisions ofbreast cancer in a Tumor Marker Utility Grading System (Hayes et al.,Breast Cancer Res. Treat. 52:305-19, 1998). A gene prognosis profile of70 genes, developed at the Netherlands Cancer Institute, has beencharacterized as a good predictor of outcome (van de Vijver et al., N.Engl. J. Med. 347:1999-2009, 2002). The likelihood of distant recurrencein breast cancer patients, who have estrogen receptor positive tumorsand no involved lymph nodes, can be defined with a panel of 21 geneproducts, amplified by RT-PCR from paraffin blocks, and correlated withthe likelihood of distant recurrence (Paik et al., N. Engl. J. Med.351:2817-2826, 2004). The selective expression of OPN-c in breastcancers, but not in healthy breasts, can provide a simpler andcomparably reliable diagnostic and prognostic marker.

The disclosed methods and kits can provide additional treatment options.For example, if a subject is positive for ER, PR or HER2, there arestandard treatment regimens (e.g., Herceptin for HER2+ or tamoxifen forER+ or PR+). However, for subjects that are negative for all three ofER, PR and HER2, there is no current method to determine how thesesubjects will progress. It is shown herein that at least 38% of subjectswho are negative for ER, PR and HER2 are highly positive for OPN-c, andthe OPN-c highly positive subjects are those having more aggressivecancers (e.g., grade 2 and 3). Therefore, the disclosure providesmethods of selecting patients having breast or other cancers that arehighly OPN-c positive for additional, more aggressive therapies (such asa mastectomy instead of a lumpectomy or a lumpectomy combined withchemotherapy (such as Herceptin® therapy if the tumor overexpresses HER2and tamoxifen or other anti-estrogen therapy if the tumor overexpressesER) instead of a lumpectomy alone).

Provided herein are methods of grading and/or staging an OPN-expressingcancer, such as breast cancer, for example DCIS or LCIS. In someexamples, the cancer is a breast cancer is known to be ER− (ERnegative), PR−, and HER2−. The method can include detecting or measuringthe OPN-c expression level in a breast or other cancer sample obtainedfrom the subject, wherein the presence of significant amounts ofdetectable OPN-c in the breast cancer or other cancer sample (e.g., top⅓ intensity or top ⅔ intensity) indicates that the subject has moreaggressive cancer (such as a higher grade, such as grade 2 or 3 breastcancer), the presence of moderate amounts of detectable OPN-c (e.g.,mid-⅓ intensity) in the breast or other cancer sample indicates that thesubject has a moderately aggressive cancer (such as a grade 2 breastcancer), while the presence of lower amounts of detectable OPN-c (e.g.,lower ⅓ intensity) in the breast or other cancer sample indicates thatthe subject has a less aggressive cancer (such as a lower grade, such asgrade 1 breast cancer). In some examples, the method further includesselecting a subject having or suspected of having breast or other cancerthat expresses OPN (such as OPN-c).

In some examples, the level of OPN-c expression is quantified or scaled,and may optionally be compared to a control or reference value (or rangeof values) of OPN-c expression (such as a positive or negative cancercontrol, or a particular grade of tumor). Quantification does notrequire determining an absolute amount, but can include determining arelative amount. For example, the relative or absolute quantity of OPN-cin a sample can be determined. In some examples, the relative amount ofOPN-c present is determined, for example by quantifying the amount ofOPN-c present. In some examples, OPN-c expression levels are normalized,for example normalized by comparing OPN-c expression to a control (e.g.,the level of expression of a housekeeping gene such as β-actin or GAPDH)in the tumor sample. In some examples, OPN-c (and in some examples othercancer markers or housekeeping genes known in the art) is also detectedor measured in normal tissue, or in non-cancerous tissue adjacent tocancer tissue. For example, normal breast tissue can serve as a negativeor background control for OPN-c, as OPN-c is not expressed in normalbreast tissue (see FIG. 1A).

In one example, immunohistochemistry (IHC) is used to detect OPN-c (andother cancer markers). In some examples when IHC is used, the scale ofdetectable staining is represented on a typical 1+ to 3+ scale torepresent the intensity, wherein 0 is assigned to negative staining, 3being assigned to very intensely staining samples, and 1 assigned toweakly staining samples (see Table 1). In some examples a value of 2 isre-tested. Therefore, in particular examples, if a level of “1” isdetected for OPN-c, this indicates that the subject has a lessaggressive cancer (such as a grade 1 breast cancer or a stage 0-I breastcancer), while detection of a level “3” for OPN-c indicates that thesubject has an aggressive cancer (such as a grade 2 or 3 breast canceror a stage II or higher breast cancer). For example, as shown in Table1, tumors can be graded depending on their relative level of OPN-cexpression. One skilled in the art will appreciate that these values arenot absolutes and can vary depending on the samples and reagents used.

TABLE 1 Exemplary correlation of OPN-c staining intensity to tumorgrade. Staining Intensity Report Result Score Microscope ObservationTumor Grade Positive: Any 3+  Strong reactivity: Dark brown to black 3IHC staining of staining is usually, but not always, in a tumor cellcomplete membrane pattern, producing membranes a thick outline of thecell. Cytoplasmic above reactivity may be absent or may be backgroundlevel moderately intense when membrane whether it is staining is veryintense. complete or Submembranous cytoplasmic incomplete accentuationmay be present. circumferential 2.5 Intense reactivity: Shades of brown2 staining in more staining of medium darkness than 0% tumor(intensity). Membranous reactivity is cells usually but not alwayscomplete, producing a circular outline of the neoplastic cell.Incomplete membrane reactivity of moderate intensity is also considered2+. The cytoplasmic reactivity is of weaker intensity than the membranereactivity. 2+  Moderate reactivity: Shades of brown 2 staining ofintermediate darkness (intensity). Membranous reactivity is usually butnot always complete, producing a circular outline of the neoplasticcell. Incomplete membrane reactivity of moderate intensity is alsoconsidered 2+. The cytoplasmic reactivity is of weaker intensity thanthe membrane reactivity. 1.5 Slight reactivity: Staining of 1intermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brown 1reactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: 0.5 Trace reactivity: Trace brown 1Absence of reactivity where membranous and membrane cytoplasmiclocalization is staining above indeterminate. background in all 0   Noreactivity tumor cells. Presence of cytoplasmic in the absence ofmembrane staining.

The methods of the present disclosure are not limited to particularmethods of representing OPN-c expression levels present in the subjectsample. Although the typical 1+ to 3+ scale to represent the signalintensity can be used (for example when IHC is used to detect a targetmolecule), other scales can be used. In one example, the mean combinedscore is used to represent the OPN-c levels present in a subject sample.The mean combined score methods can provide a scale ranging from 0 to300. The tissues are assessed by scoring the cytoplasm using a typical1+ to 3+ scale to represent the intensity, with 3 being assigned to veryintensely staining samples, 1 to weakly staining samples, and 0 tonegatively staining samples. Combined pathology scores are calculatedfrom mean intensity (ranging from 1 to 3) multiplied by mean percent(ranging from 0 to 100) of tissue stained. The highest possible score is300. To assign a tumor grade, the modified Scarff-Bloom-Richardson (SBR)system may be used, which considers nuclear grade, tubule formation, andmitotic rate. The combination of scores for these factors is applied toassign a grade of 1, 2, or 3 (or to assign a particular tumor stage). Insome examples, the signal (or value in the range of 0 to 300) obtainedfrom the sample tissue is compared to a control signal (or value orrange of values expected from a control sample). For example, theresulting value from the experimental sample in the range of 0 to 300can be compared to control samples having known tumor grades (or normaltissue) and their resulting values on the 0 to 300 sale, or can becompared to expected values on the 0 to 300 scale for each tumor grade(or stage). For example if a control grade 3 tumor has a range of 250 to300, control grade 2 tumor has a range of 150 to 249, control grade 1tumor has a range of 50 to 149, and the normal sample has a range of 0to 49, if the experimental value obtained is 275 the experimental sampleis concluded to be a grade 3 tumor while if the experimental valueobtained is 74 the experimental sample is concluded to be a grade 1tumor. One skilled in the art will appreciate that the values providedabove are for demonstration purposes, and that absolute values or rangesof values will vary, for example depending on the method used to detectexpression.

Yet other methods that can be used to represent the OPN-c levels presentin a subject sample include a continua of numbers, whose absolute valuemay vary depending on the particular experimental sample and/or theparticular control (e.g., particular housekeeping gene) used. Examplesof such methods are known in the art (see for example U.S. Pat. Nos.7,056,674 and 7,081,340; herein incorporated by reference). For example,if nucleic acid detection methods are used, for example real timeRT-PCR, OPN-c and a control (e.g., a housekeeping gene) are detected inthe experimental sample. Using this information, OPN-c expression can benormalized against the expression level of a housekeeping transcript,for example by generating a ratio of OPN-c expression to controlexpression. The normalization can be used to correct for differences inthe amount of RNA assayed and variability in the quality of the RNAused. In some examples, the method also includes detecting expression ofa normalizing gene, such as a housekeeping gene. Alternatively,normalization can be based on the mean or median signal of all of theassayed genes or a large subset thereof (global normalization approach).On a gene-by-gene basis, measured normalized amount of a patient tumormRNA can be compared to the amount found in a breast cancer tissuereference set. The number of breast cancer tissues in this reference setshould be sufficiently high to ensure that different reference sets (asa whole) behave essentially the same way. Typically, the breast cancertissue reference set consists of at least about 30 different breastcancer tissue specimens. The level measured in a particular tumor sampleto be analyzed falls at some percentile within this range, which can bedetermined by methods well known in the art.

The higher the OPN-c:housekeeping gene expression level ratio for aparticular housekeeping gene, the higher the grade of cancer in theexperimental sample (e.g., grade 3), while a lower OPN-c:housekeepinggene expression level ratio for a particular housekeeping gene indicatesa lower grade of cancer. One skilled in the art will appreciate that theabsolute ratio for a particular OPN-c:housekeeping gene and thecorresponding grade (or stage) of tumor, may vary. For example, if acontrol grade 3 tumor has a OPN-c:β-actin ratio of greater than 3,000,control grade 2 tumor has a OPN-c:β-actin ratio of 500 to 2,999, controlgrade 1 tumor has a OPN-c:β-actin ratio of 50 to 499, and the normalsample has a OPN-c:β-actin ratio of less than 50, if the experimentalOPN-c:β-actin ratio obtained is 9,000 the experimental sample isconcluded to be a grade 3 tumor while if the experimental OPN-c:β-actinratio obtained is 1,000 the experimental sample is concluded to be agrade 1 tumor. On the other hand, if a different housekeeping gene isused, such as GAPDH, the range of values may differ. For example, Forexample, if a control grade 3 tumor has a OPN-c:GAPDH ratio greater than10,000, control grade 2 tumor has a OPN-c:GAPDH ratio of 1000 to 9,999,control grade 1 tumor has a OPN-c:GAPDH ratio of 999 to 100, and thenormal sample has a OPN-c:GAPDH ratio of less than 100, if theexperimental OPN-c:GAPDH ratio obtained is 60,000 the experimentalsample is concluded to be a grade 3 tumor while if the experimentalOPN-c:GAPDH ratio obtained is 500 the experimental sample is concludedto be a grade 1 tumor. One skilled in the art will appreciate that thevalues provided are for demonstration purposes, and that absolute valuesor ranges of values will vary, for example depending on the method usedto detect expression and the particular housekeeping gene detected.

The method can further include detecting or measuring other cancermarkers in the cancer sample. For example, if the sample is a breastcancer marker, the method can include detecting or measuring one or moreof estrogen receptor (ER); progesterone receptor (PR); and humanepidermal growth factor receptor 2 (HER2). In some examples, ER, PR andHER2 are detected. In some examples, the read-out for ER and PR issimply positive or negative, and the read-out for HER2 is on a scale asdescribed above for OPN-c (e.g., see Table 1). In some examples, theabsence of ER (ER−) and PR (PR−) and the presence of OPN-c and HER2 inthe breast cancer sample indicates that the subject has a moreaggressive breast cancer (e.g., grade 2 or 3 breast cancer), while thepresence of ER (ER+), PR (PR+), and lower HER2 (e.g., 0 or 1), and OPN-c(e.g., 1) in the breast cancer sample indicates that the subject has aless aggressive form of breast cancer (e.g., grade 1 breast cancer). Insome examples, the presence of significant HER2 (e.g., 3 on a scale of 0to 3) and OPN-c (e.g., 3 on a scale of 0 to 3) in the breast cancersample indicates that the subject has a more aggressive breast cancer(e.g., grade 2 or 3 breast cancer). To assess the predictive value ofseveral biomarkers in combination (e.g., OPN-c, ER, PR and HER2), alogistic regression modeling approach can be used, which assesses amongseveral models the one that performs best. For the best performingmodel, a ROC (receiver operating characteristic) curve can be generatedas a graphical plot of sensitivity/specificity as the discriminationthreshold is varied.

The OPN-c, ER, PR, and HER2 molecules (as well as housekeeping genes andother cancer markers) can be detected by detecting proteins or nucleicacid molecules. For example, antibodies can be used to detect OPN-c, ER,PR, and HER2 proteins, and nucleic acid probes or primers can be used todetect OPN-c, ER, PR, and HER2 nucleic acid molecules. Methods of suchdetection are routine, and include immunohistological methods, Westernblotting, and flow cytometry, mass spectrometry, as well as PCR andnucleic acid arrays. In some examples, the antibody or nucleic acidprobe/primer includes a detectable label, such as a fluorophore, topermit detection of the antibody. In some examples, the antibody isdetected with an appropriately labeled secondary antibody.

In some examples, detection of both OPN-c protein and OPN-c nucleic acidmolecules in one or more biological samples obtained from the subject(such as a breast cancer tissue sample) are used to grade or stage atumor that overexpresses OPN-c (such as DCIS).

In some examples, the method further includes selecting a treatmentprotocol for the subject based on the OPN-c detection. For example, ifthe cancer sample is highly positive for OPN-c (and in some examplesnegative for ER, PR, and HER2), a more aggressive therapeutic protocolcan be selected and administered, as this indicates the subject has amore aggressive form of cancer (e.g., grade 3). Exemplary moreaggressive therapies include a mastectomy instead of a lumpectomy, alumpectomy combined with radiotherapy and/or chemotherapy (e.g.,anti-HER2, anti-ER, and/or anti-PR therapies for example Trastuzumab(Herceptin®), bevacizumab (Avastin®), pertuzumab (OmniTarg™), ZM105180(Zemab®), ertumaxomab (Rexonum), Arimidex® and tamoxifen) instead oflumpectomy alone, or an increase in the dose and/or number of courses ofchemotherapy. Methods of administering such therapies are routine in theart and can be designed by skilled clinicians. In contrast, if thecancer sample has lower amounts of OPN-c (and in some examples negativefor ER, PR, and HER2), a less aggressive therapeutic protocol can beselected and administered, as this indicates the subject has a lessaggressive form of cancer (e.g., grade 1). Exemplary less aggressivetherapies include a lumpectomy instead of a mastectomy, a lumpectomyalone instead of a lumpectomy combined with radiotherapy and/orchemotherapy, or a decrease in the dose and/or number of courses ofchemotherapy. Methods of administering such therapies are routine in theart and can be designed by skilled clinicians.

In some examples, the diagnostic antibodies for ER, PR, HER-2 areobtained from Ventana Medical Systems (Tuscon, Ariz.). However, oneskilled in the art will appreciate that other antibodies that can beused in the methods and kits provided herein are commercially availablefrom other sources, such as: Novus Biologicals (Littleton, Colo.), SantaCruz biotechnology, Inc. (Santa Cruz, Calif.), and Invitrogen (Carlsbad,Calif.). In some examples, the OPN-c antibody is the one described inExample 1. However, one skilled in the art will appreciate that otherOPN-c antibodies that can be used in the methods and kits providedherein are commercially available from other sources, such as GallusImmunotech, Inc. (Ontario, Canada).

Biological Samples

Exemplary samples include, without limitation, blood smears,cytocentrifuge preparations, cytology smears, core biopsies, fine-needleaspirates, and/or tissue sections (e.g., cryostat tissue sections and/orparaffin-embedded tissue sections). Methods of obtaining a biologicalsample from a subject are known in the art. For example, methods ofobtaining breast tissue or breast cells are routine. Exemplarybiological samples may be isolated from normal cells or tissues, or fromneoplastic cells or tissues. Neoplasia is a biological condition inwhich one or more cells have undergone characteristic anaplasia withloss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and which cells may be capable of metastasis.Exemplary neoplastic cells or tissues may be isolated from solid tumors,including breast carcinomas (e.g. lobular and duct carcinomas),adrenocortical cancer, ameloblastoma, ampullary cancer, bladder cancer,bone cancer, cervical cancer, cholangioma, colorectal cancer,endometrial cancer, esophageal cancer, gastric cancer, glioma, granularcall tumor, head and neck cancer, hepatocellular cancer, hydatiformmole, lung cancer, lymphoma, melanoma, mesothelioma, myeloma,neuroblastoma, oral cancer, osteochondroma, osteosarcoma, ovariancancer, pancreatic cancer, pilomatricoma, prostate cancer, renal cellcancer, salivary gland tumor, soft tissue tumors, Spitz naevus, squamouscell cancer, teratoid cancer, and thyroid cancer.

For example, a sample from a tumor that contains cellular material canbe obtained by surgical excision of all or part of the tumor, bycollecting a fine needle aspirate from the tumor, as well as othermethods known in the art. If desired, the sample can be concentrated orpurified before use. For example, proteins or nucleic acids can beisolated from the sample. Such methods are routine in the art.Alternatively, the sample can be used directly. In particular examples,a tissue or cell sample is applied to a substrate and analyzed todetermine if it contains detectable levels of OPN-c (and in someexamples also ER, PR, and HER2). A solid support useful in a disclosedmethod need only bear the biological sample and, optionally, butadvantageously, permit the convenient detection of components (e.g.,proteins and/or nucleic acid sequences) in the sample. Exemplarysupports include microscope slides (e.g., glass microscope slides orplastic microscope slides), coverslips (e.g., glass coverslips orplastic coverslips), tissue culture dishes, multi-well plates, membranes(e.g., nitrocellulose or polyvinylidene fluoride (PVDF)) or BIACORE™chips. In particular examples, a breast cancer sample obtained from thesubject is analyzed to determine if it contains detectable levels ofOPN-c mRNA or protein.

Fixatives for mounted cell and tissue preparations are well known in theart and include, without limitation, 95% alcoholic Bouin's fixative; 95%alcohol fixative; B5 fixative, Bouin's fixative, formalin fixative,Karnovsky's fixative (glutaraldehyde), Hartman's fixative, Hollande'sfixative, Orth's solution (dichromate fixative), and Zenker's fixative(see, e.g., Carson, Histotechology: A Self-Instructional Text,Chicago:ASCP Press, 1997).

Detection of Peptides

In particular examples, a sample obtained from the subject is analyzedto determine if it contains detectable levels of OPN-c protein. In someexamples, the sample is a breast cancer sample. The sample, for examplea breast cancer sample, can also be analyzed for the presence of ER, PR,and HER2 proteins.

Methods of detecting proteins are routine. In some examples,immunoassays are used to detect the presence of OPN-c protein in thesample (and in some examples additionally one or more of ER, PR, andHER2 protein). Generally, immunoassays include the use of one or morespecific binding agents (such as antibodies) that can substantially onlybind to the target peptide, such as OPN-c, ER, PR, and HER2. Suchbinding agents can include a detectable label (such as a radiolabel,fluorophore or enzyme), that permits detection of the binding to theprotein. Exemplary immunoassays that can be used include, but are notlimited to: Western blotting, ELISA, fluorescence microscopy, and flowcytometry. A particular immunoassay is immunohistochemistry.

In one example, the specific binding agent is an antibody, such as apolyclonal or monoclonal antibody, or fragment thereof. In someexamples, the antibody is a humanized antibody. In some examples, theantibody is a chimeric antibody. If desired, the antibody can include adetectable label to permit detection and in some cases quantification ofthe target protein/antibody complex.

The presence of detectable signal above background or control levelsindicates that the presence of a target peptide (e.g., OPN-c and in someexamples additionally one or more of ER, PR, and HER2 protein) in thesample. For example, the level of OPN-c detected can be compared to acontrol or reference value (or range of values), such as a value thatrepresents a level of OPN-c protein expected if a breast cancer is agrade 3, a value that represents a level of OPN-c protein expected if abreast cancer is a grade 2, a value that represents a level of OPN-cprotein expected if a breast cancer is a grade 1, a value thatrepresents a level of OPN-c protein expected if no breast cancer ispresent (normal tissue), or combinations thereof. Similar referencevalues can be used for ER, PR, and HER2 proteins. In some examples, thecontrol is a normal (e.g., non-tumor) sample (or value expected for anormal sample).

In some examples, detection of higher levels of OPN-c indicates thatthat subject has a grade 3 tumor, detection of intermediate levels ofOPN-c indicates that that subject has a grade 2 tumor, while detectionof lower levels of OPN-c indicates that that subject has a grade 1tumor. In some examples, detection of higher levels of OPN-c indicatesthat that subject has a more aggressive tumor (such as stage III orabove), detection of intermediate levels of OPN-c indicates that thatsubject has a moderately aggressive tumor (such as a stage II tumor),while detection of lower levels of OPN-c indicates that that subject hasa less aggressive tumor (such as a stage I or less). The amount of OPN-cdetected can depend on the measurement used (e.g., see FIG. 3C).Detection of OPN-c or other cancer marker (e.g., PR, ER, HER2) can berepresented as the mean cytoplasmic combined score, mean percentpositive, or mean percent intensity, or other value known in the art.The value obtained for the test cancer sample can be compared to areference value (or range of values), such as a reference valuerepresenting a value or range of values expected for grade 1, 2, or 3breast cancer (or a particular stage of cancer). In some examples, thereference is a sample possessing a known or expected amount of OPN-cprotein. For example, cell lines MCF-7 and ZR-75 are negative for OPN-c,while MDA-MB-435 and MDA-MB-231 are positive for OPN-c (all cell linesavailable from American Type Culture Collection). In some examples, thesample is one having a known grade 1, 2, or 3 breast cancer and aparticular value or range of values for OPN-c (and in some examples alsovalues for ER, PR, and HER2). The test value for OPN-c (and in someexamples also test values for ER, PR, and HER2) can be compared to thereference value, to correlate the value to a grade of cancer. Forexample, if the level of OPN-c detected in the subject's sample issimilar or greater than the level of OPN-c in a known grade 3 breastcancer sample or falls in the range expected for a grade 3 breastcancer, this indicates that the subject has a grade 3 breast cancer.

Detection of Nucleic Acid Molecules

In particular examples, a sample obtained from the subject is analyzedto determine if it contains detectable levels of OPN-c nucleic acidmolecules (and in some examples also ER, PR, and HER2), such as a breastcancer sample, a sample adjacent to the tumor, normal breast tissue, orcombinations thereof. In particular examples, OPN-c (and in someexamples also ER, PR, and HER2) nucleic acid molecules (such as mRNA orcDNA) are measured.

Methods of detecting nucleic acid molecules are routine. In particularexamples, a breast cancer sample obtained from the subject is analyzedto detect OPN-c nucleic acid molecules (and in some examples also ER,PR, and HER2 nucleic acid molecules), such as cDNA or mRNA. For example,assays that permit detection of nucleic acids can be used. Exemplaryassays that can be used include, but are not limited to: Northernblotting, Southern blotting, PCR (such as RT-PCR or real-time RT-PCR),and DNA arrays. For example, OPN-c can be amplified from a sample usingPCR, and the OPN-c amplicons detected and in some examples quantified.

In some examples, detection of higher levels of OPN-c cDNA or mRNAindicates that that subject has a grade 3 tumor, detection ofintermediate levels of OPN-c cDNA or mRNA indicates that that subjecthas a grade 2 tumor, while detection of lower levels of OPN-c cDNA ormRNA indicates that that subject has a grade 1 tumor. In some examples,detection of higher levels of OPN-c cDNA or mRNA indicates that thatsubject has a more aggressive tumor (such as stage III or above),detection of intermediate levels of OPN-c cDNA or mRNA indicates thatthat subject has a moderately aggressive tumor (such as a stage IItumor), while detection of lower levels of OPN-c cDNA or mRNA indicatesthat that subject has a less aggressive tumor (such as a stage I orless). The amount of OPN-c cDNA or mRNA detected can depend on themeasurement used.

In one example, a nucleic acid probe that hybridizes to an OPN-c nucleicacid is contacted with the breast or other cancer cancer sample. Forexample, the probe can be incubated with the sample under highstringency conditions (such as when the hybridization is performed atabout 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4),5×SSC, 5× Denhart's solution, 50 μg/mL denatured, sonicated salmon spermDNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about5×10⁷ cpm/μg), while the washes are performed at about 65° C. with awash solution containing 0.2×SSC and 0.1% sodium dodecyl sulfate),wherein the presence of higher amounts of signal indicates that thebreast cancer is a grade 3 cancer and the presence of lower amounts ofsignal indicates that the breast cancer is a grade 1 cancer.

Real-time RT-PCR reactions can be performed using routine methods in theart. For example, a SYBR Green detection format can be used withoptimized concentrations of template, primers, and MgCl₂. For each testsample, a no-template reaction can be included as a negative control.The reaction conditions can include 35-40 cycles of melting, a primerset specific annealing temperature, extension, and melting curveprogram, and finally a cooling step. Product purity, product size, andabsence of primer dimers can be confirmed by DNA melting curve analysisand agarose gel electrophoresis. The threshold value for amplificationcan compared to a housekeeping gene and to a reference sample. Therelative abundance of the RNA message is then calculated by methodsknown to the skilled artisan (see e.g., Pfaffl Nucleic Acids Res. 2001;29:e45, herein incorporated by reference as to the method).

The value obtained (or signal detected) for the test breast cancersample can be compared to a reference value, such as a reference valuerepresenting a value or range of values expected for grade 1, 2, or 3breast cancer (or a particular stage of cancer). In some examples, thereference is a sample possessing a known or expected amount of OPN-cnucleic acid molecule. For example, cell lines MCF-7 and ZR-75 arenegative for OPN-c, while MDA-MB-435 and MDA-MB-231 are positive forOPN-c (all cell lines available from American Type Culture Collection).In some examples, the sample is one having a known grade 1, 2, or 3breast cancer and a particular value or range of values for OPN-cnucleic acid molecules (and in some examples also values for ER, PR, andHER2). The test value for OPN-c (and in some examples also test valuesfor ER, PR, and HER2) can be compared to the reference value, tocorrelate the value to a grade of cancer. For example, if the level ofOPN-c nucleic acid molecule detected in the subject's sample is similaror greater than the level of OPN-c nucleic acid molecule in a knowngrade 3 breast cancer sample or falls in the range expected for a grade3 breast cancer, this indicates that the subject has a grade 3 breastcancer.

Methods of Prognosing Cancer

Methods are provided herein for prognosing a cancer by examining OPN-clevels in a tumor sample obtained from a subject. OPN-c can be detectedas described above, wherein the presence of significant amounts ofdetectable OPN-c in the cancer sample (e.g., top ⅓ intensity or top ⅔intensity) indicates that the subject has a worse prognosis while thepresence of lower amounts of detectable OPN-c (e.g., lower ⅓ intensity)in the cancer sample indicates that the subject has a better prognosis.Detection of OPN-c in a tumor sample can be used to prognose anyOPN-expressing tumor, such as adrenocortical cancer, ameloblastoma,ampullary cancer, bladder cancer, bone cancer, breast cancer, cervicalcancer, cholangioma, colorectal cancer, endometrial cancer, esophagealcancer, gastric cancer, glioma, granular call tumor, head and neckcancer, hepatocellular cancer, hydatiform mole, lung cancer, lymphoma,melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer,osteochondroma, osteosarcoma, ovarian cancer, pancreatic cancer,pilomatricoma, prostate cancer, renal cell cancer, salivary gland tumor,soft tissue tumors, Spitz naevus, squamous cell cancer, teratoid cancer,and thyroid cancer.

Prognostic methods can be used to predict likely tumor progression,survival time, likely worsening of the condition, and the like, forexample the prognosis in the absence of therapy (e.g., chemotherapy orradiation therapy). For example, the methods can be used to determinewhether a tumor may be less aggressive (e.g., less rapidly growing,and/or less likely to metastasize) or more aggressive (e.g., rapidlygrowing, and/or more likely to metastasize), for example independent oftherapy. A less-aggressive tumor can be characterized by any parametersknown in the art, including, for instance, decreased growth rate (e.g.,increased rate of apoptosis and/or decreased rate of cell division),decreased rate of metastasis, and/or increased sensitivity tochemotherapy. A more aggressive tumor can be characterized by anyparameters known in the art, including, for instance, increased growthrate (e.g., decreased rate of apoptosis and/or increased rate of celldivision), increased rate of metastasis, and/or decreased sensitivity tochemotherapy. In some examples, prognosis for a subject can becharacterized by actual survival after initial diagnosis (such as6-month survival, 1-year survival, 2-year survival, or 5-year survival),and/or actual survival relative to the average survival for similarlysituated patients. A better prognosis entails, e.g., survival of apatient for more than 1 year after initial diagnosis (such as more than2 years or more than 5 years), or survival of a patient for more than 6months longer (e.g., more than 1 year longer, more than 2 years longer,more than 5 years longer) than the average survival for similarlysituated. A worse prognosis entails, e.g., survival of a patient forless than 5 years after initial diagnosis (such as less than 2 years orless than 1 years), or survival of patient less than the averagesurvival for similarly situated patients (such as, about 3 months lessthan average survive, about 6 months less than average survive, or about1 year less than average survival).

As described above, in some examples, the amount of OPN-c is quantifiedor scaled, wherein higher levels of OPN-c indicate a more aggressivecancer and a worse prognosis, and lower levels of OPN-c indicate a lessaggressive cancer and better prognosis. For example, if the typical 1+to 3+ scale is to represent the intensity as described above, if a levelof “1” is detected for OPN-c, this indicates that the subject has a lessaggressive cancer (e.g., decreased likelihood of death within 5 years ofdiagnosis or decreased likelihood of metastasis), while detection of alevel “3” for OPN-c indicates that the subject has an aggressive cancer(e.g., increased likelihood of death within 5 years of diagnosis orincreased likelihood of metastasis). In other examples, the meancombined score method is used as described above (where scores rangefrom 0 to 300). For example, the resulting value from the experimentalsample in the range of 0 to 300 can be compared to control sampleshaving known prognosis and values on the 0 to 300 sale, or can becompared to expected values on the 0 to 300 scale for each prognosis.For example if a control poor prognosis (e.g., 5 year survival rate ofless than 5%) has a range of 250 to 300, and a control good prognosis(e.g., 5 year survival rate of greater than 95%) has a range of 0 to 49,if the experimental value obtained is 275 the experimental sample isconcluded to have a poor prognosis while if the experimental valueobtained is 20 the experimental sample is concluded to have a goodprognosis. One skilled in the art will appreciate that the valuesprovided above are for demonstration purposes, and that absolute valuesor ranges of values will vary, for example depending on the method usedto detect expression.

In other examples, OPN-c nucleic acid levels present in a subject sampleare normalized as described above (see for example U.S. Pat. Nos.7,056,674 and 7,081,340; herein incorporated by reference). Inparticular examples, the higher the ratio for a particular housekeepinggene, the worse the prognosis, while a lower ratio for a particularhousekeeping gene indicates a better prognosis.

Kits

The present disclosure provides kits that can be used to grade, stage,and prognose abreast or other OPN-expressing cancer, for example todetermine if a cancer is more or less aggressive, determine thelikelihood that a cancer will metastasize, and predicting the survivaltime of a subject with cancer. Generally cancer grading is performed ona scale of 1 to 3, with 1 being less aggressive and 3 being the mostaggressive.

In particular examples, the kit includes or consists of an antibody thatspecifically binds estrogen receptor (ER); an antibody that specificallybinds progesterone receptor (PR); an antibody that specifically bindsHER2; and an antibody that specifically binds OPN-c. In some examples,the kit does not include a PR antibody. In some examples, the kitincludes or consists of a nucleic acid probe or primer specific for anER nucleic acid sequence; a nucleic acid probe or primer specific for aPR nucleic acid sequence; nucleic acid probe or primer specific for aHER2 nucleic acid sequence 2; and nucleic acid probe or primer specificfor an OPN-c nucleic acid sequence. In particular examples, the kit doesnot include a PR nucleic acid probe or primer. In some examples, theantibody or nucleic acid probe or primer is labeled (for example, with afluorescent, radioactive, or an enzymatic label).

In some embodiments, the antibodies, probes, or primers are providedsuspended in an aqueous solution or as a freeze-dried or lyophilizedpowder, for instance. The container(s) in which the antibodies, probes,or primers are supplied can be any conventional container that iscapable of holding the supplied form, for instance, microfuge tubes,ampoules, or bottles. The amount of antibodies, probes, or primerssupplied can be any appropriate amount, such as from about 1 to about 5μg/ml.

In other embodiments, control slides upon which are mounted one or moretissue or cell preparations (e.g., xenografts, cell pellets, or clottedcells) that may serve as positive and/or negative controls for OPN-c,ER, PR, and/or HER2 expression may be provided in an appropriate andseparate container.

The kits can include antibodies or nucleic acid molecules specific forone or more housekeeping molecules, such as one or more of GAPDH(glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinatedehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L(HBS1-like protein), β-actin, and AHSP (alpha haemoglobin stabilizingprotein).

The kit can also include one or more control samples, such as positiveor negative control samples. For example, a controls sample can be anormal breast or breast cancer sample with known relative amounts of ER,PR, HER2 and OPN-c. In a particular example, the control sample is axenograft control.

One skilled in the art will appreciate that the kits can include otheragents to facilitate the particular application for which the kit isdesigned. For example, the kit can additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like), as well as buffers and otherreagents routinely used for the practice of a particular diagnosticmethod.

Example 1 Materials and Methods

This example describes the materials and methods used to obtain theresults described in Examples 2-5. Although particular exemplary methodsand materials are described, one skilled in the art will appreciate thatvariations can be made. For example, different antibodies can be used.

Human breast cancer RNA: Specimens of human breast tumors,non-transformed surrounding tissue, as well as healthy breast tissue(obtained from reduction mammoplasties) were provided by the tissueprocurement facility of the University of Cincinnati MedicalCenter/Children's Hospital. Samples were obtained from 20 invasiveductal carcinomas at grades The average patient age was 55 years,ranging from 35 to 84 years. The tumor stages ranged from T1 to T4 andfrom N0 to N4, the status of metastases was unknown in all cases. Elevensamples of tumor-surrounding tissue came from patients with the mean ageof 61 years, ranging from 46 to 84 years. Normal tissues were obtainedfrom 22 reduction mammoplasties. The patient age ranged from 21 to 57,with an average of 36.5 years. Total RNA was extracted from specimensusing TRIZOL® Reagent (Invitrogen). Total RNA was used for cDNAsynthesis by reverse transcription using Superscript II (Invitrogen)according to the manufacturer's protocol in a total volume of 20 μl.

PCR Amplification: All PCR reactions were performed on a Cepheid(Sunnyvale, Calif.) Smart Cycler using SYBR Green detection format. 0.5μl of cDNA was added to each PCR reaction in a total volume of 25 μlusing the standard Invitrogen PCR buffer system with optimizedconcentrations of MgCl₂. For each experiment a no-template reaction wasincluded as a negative control. The conditions for PCR were 94° C.denaturation for 120 s followed by 35-40 cycles of: 94° C. melting for15 s, a primer set specific annealing temperature (Table 2), extensionat 72° C. and melting curve program (60-95° C. with a heating rate of0.2° C. per second and a continuous fluorescence measurement), andfinally a cooling step to 4° C. Product purity, product size, andabsence of primer dimers were confirmed by DNA melting curve analysisand agarose gel electrophoresis. Melt curves yielded a single sharp peakfor all template reactions, and a minimal melt peak (resulting fromprimer dimers) or no melt peaks for the no-template control reactions.

TABLE 2 Primer pairs for real-time RT-PCR. Target Product [Mg²⁺] Anneal-sequence Primer Sequence (SEQ ID NO:) size (bp) (mM) ing (° C.) OPN-aforward 5′-ATCTCCTAGCCCCACAGAAT-3′ (1) 208 2.5 58 reverse5′-CATCAGACTGGTGAGAATCATC-3′ (2) OPN-b forward 5′-ATCTCCTAGCCCCAGAGAC-3′(3) 209 2.5 62 reverse 5′-AAAATCAGTGACCAGTTCATCAG-3′ (4) OPN-c forward5′-TGAGGAAAAGCAGAATGCTG-3′ (5) 155 3.0 62 reverse5′-GTCAATGGAGTCCTGGCTGT-3′ (6) β-Actin forward5′-GGCGGCACCACCATGTACCCT-3′ (7) 200 2.0 65 reverse5′-AGGGGCCGGACTCGTCATACT-3′ (8) Ck-19 forward 5′-CCCGCGACTACAGCCACTA-3′(9) 163 2.0 60 reverse 5′-CTCATGCGCAGAGCCTGTT-3′ (10) aP2 forward5′-TCAGTGTGAATGGGGATGTG-3′ (11) 249 1.6 58 reverse5′-GTGGAAGTGACGCCTTTCAT-3′ (12) Shown are the primer sequences and thesizes of the resulting PCR products. The magnesium concentrations andannealing temperatures are indicated as optimized in preliminaryexperiments. The primer sets for osteopontin (OPN) were designed todistinguish the splice variants. The primer sets for CK-19 (Benoy etal., Brit. J. Canc. 91: 1813-20, 2004), β-actin and aP-2 covered twodifferent exons to avoid amplification of any contaminating genomic DNAAmplification efficiencies were determined for each given primer set bycDNA dose-response curve analysis.

For conventional RT-PCR, the osteopontin splice variants were amplifiedwith specific primer pairs [14, herein incorporated by reference as tothe primers]. The amplification of GAPDH with primers5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ (forward; SEQ ID NO: 13) and5′-CATGTGGGCCATGAGGTCCACCAC-3′ (reverse; SEQ ID NO: 14) served as acontrol for equal loading and integrity of the cDNA. All PCR productswere analyzed by Tris-acetate EDTA agarose (2.5% w/v) gelelectrophoresis.

Immunohistochemistry: A normal breast multi-array (Cybrdi, Fredrick,Md.) containing 69 normal cores (plus three infiltrating ductalcarcinomas as references) and a breast carcinoma multi-array containing56 cancer cores confirmed by the examining pathologist (7 cores werediagnosed not to contain carcinoma and were therefore excluded), werestained for OPN-c. The carcinoma array included intraductal carcinomas,infiltrating ductal carcinomas, infiltrating lobular carcinomas,papillary adenocarcinomas, mucinous adenocarcinomas, and Paget'sdisease.

The immunohistochemistry analyses were conducted on the automatedDiscovery™ or Benchmark™ staining platform (Ventana Medical Systems,Tucson, Ariz.). Antigen retrieval for osteopontin-c was not required.The slides were incubated with an affinity-purified anti-OPN-c chickenIgY (produced by Gallus Immunotech), at 1:160 dilution for 32 minutes atroom temperature. The secondary antibody, HRP-conjugated goatanti-chicken IgY secondary antibody (Jackson ImmunoResearchLaboratories, West Grove, Pa.) diluted to 1:80 in Ventana Antibodydiluent (devoid of casein and azide), was applied for 16 minutes at roomtemperature. Antibody binding was detected using the ChromoMap™ DABdetection kit (Ventana Medical Systems). Rabbit monoclonal antibodies tohuman estrogen receptor (ER, clone SP1), progesterone receptor (PR,clone 1E2), and to HER2 (clone 4B5) from VMSI were also used to stainthe breast carcinoma array, and were detected using the UltraView™ DABkit (VMSI). The primary antibody was applied for 16 minutes at 37° C.and the HRP-conjugated anti-rabbit secondary antibody was applied for 8minutes at 37° C. All slides were counterstained with Hematoxylin II(VMSI) for 8 minutes followed by Bluing Reagent (VMSI) for 4 minutes atroom temperature.

The tissue arrays were assessed by a board-certified pathologist. Thecytoplasm was scored using a typical 1+ to 3+ scale to represent theintensity, with 3 being assigned to very intensely staining samples, 1to weakly staining samples, and 0 to negatively staining samples.Combined pathology scores were calculated from mean intensity (rangingfrom 1 to 3) multiplied by mean percent (ranging from 0 to 100) oftissue stained for the individual samples of the breast arrays. Thehighest possible score is 300. To assign a tumor grade, the modifiedScarff-Bloom-Richardson (SBR) system [15] was used, which considersnuclear grade, tubule formation, and mitotic rate. The combination ofscores for these factors was applied to assign a grade of 1, 2, or 3.

DNA constructs and transfection: The constructs for expression of thehuman osteopontin splice variants were obtained by reversetranscription-PCR from the malignant breast tumor cell line MDA-MB-435.The coding sequence of osteopontin was amplified with the primers 5′-CAAACG CCG ACC AAG GGA AAA C-3′ (SEQ ID NO: 15) and 5′-CTT CTT TCT CAG TTTATT GGT-3′ (SEQ ID NO: 16). The amplified product was TA cloned, excisedwith Xho1 and NheI, and was subcloned into the vector pCR3.1 (InvitrogenCarlsbad, Calif.). Genes cloned into this vector are expressed under thecontrol of the CMV promoter. Sequence fidelity and accurate readingframe were verified by DNA sequencing analysis. MCF-7 cells weretransfected by the Fugene method and stable clones were selected inG418.

Immunoblot assay: For the analysis of secreted osteopontin, serum-freecell culture supernatant was collected from each transfectant. 40 μl ofsupernatant per sample were electrophoresed on 10% SDS-polyacrylamidemini-gels with non-reducing sample buffer. For the analysis ofintracellular osteopontin, the cells were lysed in RIPA buffer (50 mMTris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1%sodium dodecyl sulfate). Cell lysates at equal amounts of protein (20μg/lane) were electrophoresed on reducing 10% SDS-polyacrylamide gels.The separated proteins were transferred to PVDF membranes and probedwith antibody 0-17 (Assay Designs Inc.) to osteopontin.

Example 2 Osteopontin-c RNA is Expressed in Breast Cancer but not inNormal Breast Tissue

This example describes results of measuring osteopontin gene expressionin breast cancer specimens, tumor-surrounding tissues, and normalsamples by real-time RT-PCR using splice variant specific primers andthe amplification of β-actin as a control for abundance.

Because breasts contain various amounts of fat and the source ofosteopontin are selectively the epithelial cells, the levels ofcytokeratin-19 (CK-19, epithelial marker) and aP2 (adipocyte marker)were measured and the results adjusted to account for the fraction ofepithelial cells. As shown in FIGS. 1A and 1B, OPN-c was detected in 16of 20 tumors and the RNA levels had a relative abundance comparable tothe full length form osteopontin-a. In contrast, there was no detectableOPN-c in any of the normal tissues examined. Seven of eleventumor-surrounding normal tissues were devoid of OPN-c. It is not knownwhether the four normal specimens that showed OPN-c expression hadcryptic tumor cell infiltration or a reaction to the tumor, such asinflammation. All four were grade 3 tumors without Paget disease, buttheir tumor stages varied.

The expression levels of OPN-a varied in 21 specimens of normal breasts,with only four samples lacking detectable levels (FIGS. 1A and 1B). Thismay reflect the production of unspliced osteopontin, which isphysiologically secreted in milk, by breast cells depending on thestages of the estrous cycle. The splice variant OPN-b was present at lowlevels in 18 of 20 cancers, in eight of eleven tumor surroundingtissues, and in six of 22 normal breasts (FIGS. 1A and 1B).

Statistical evaluations were performed on the data from the real timeRT-PCR measurements. According to the analysis of variance (ANOVA) onranks, the levels of osteopontin-c in the tumors are different (p<0.05)from the levels of OPN-c in the normal samples. The tumor-surroundingtissues have intermediate values and are not different from either thetumors or the normal samples. Analogous results were obtained for OPN-b.For osteopontin-a, no differences were detected among the three groupsof samples. The proportion of positives for OPN-c RNA were comparedamong the groups by the chi-square test (χ²=13.33, p<0.001), and theproportion of positives differed between normal breasts and breastcancers (p<0.0007 by Fisher's exact test), while it was notsignificantly different between tumors and surrounding normal tissues(p=0.16 by Fisher's exact test).

Every PCR run included a negative control without a template and a knowncDNA reference sample as a positive control. For the osteopontin splicevariants and β-actin, the positive control was the MDA-MB-435 breastcancer cell line that produces large amounts of RNA for all threeosteopontin forms. The positive control for CK-19 was the breast tumorcell line MCF-7, while a fatty breast specimen provided the referencefor aP2. OPN-a and β-actin levels were highly reproducible in thesesamples. The range encountered for the other amplicons also indicatedgood reproducibility, but was wider. The exact quantification of lowabundance RNA messages is inherently more subject to fluctuations thanhigher abundance messages (FIG. 1C). In sum, with the inclusion ofsuitable controls, the accuracy of these measurements will allowdiagnostic or prognostic decisions to be based on them.

Example 3 The Levels of OPN-c Correlate with Tumor Grade in Histology

An IgY antibody was raised to the splice junction of OPN-c. The antibodyselectively recognized OPN-c, but not OPN-a on Western blots (FIG. 2A).In a solid-phase ELISA, the antibody reacted strongly with GST-OPNc, butonly weakly with GST-OPNa, whereas the pan-osteopontin antibody 0-17(Assay Designs Inc.) showed very strong reactivity with both GST-OPNforms.

Immunocytochemistry was performed on cell lines with previouslyconfirmed distinct osteopontin expression levels [14]. Consistently,strong cytoplasmic staining was seen in MDA-MB-435 cells, but not inMCF-7 cells or ZR-75 cells (FIG. 2B). Staining of breast cancer tissuewith the anti-hOPNc antibody was compared to the anti-pan-osteopontinantibody 0-17. The cancer was strongly positive for total osteopontinand for OPN-c. In the absence of the respective primary antibodies, nostaining was observed (FIG. 2C).

The generated OPN-C antibody was used to analyze breast tissue arrays byimmunohistochemistry. As shown in FIG. 3A, three of 69 normal breastshad low positive staining, while all the others were negative. Among thebreast carcinomas, 43 of 56 cores (77%) stained positive for OPN-c while13 tissue samples were negative. When analyzed for its correlation withtumor grade, OPN-c staining increased from grade 1 to grade 3 (FIG. 3B),regardless of whether mean intensity, mean percent positive, or the meancytoplasmic combined score was used as a mode of assessment (FIG. 3C).

Example 4 OPN-c is a More Sensitive Marker for Breast Cancer than ER,PR, or HER2

The growth factor receptors estrogen receptor (ER), progesteronereceptor (PR) and HER2 may be up-regulated in breast cancer and are usedin diagnosis to facilitate therapy decisions. Therefore OPN-c expressionwas compared to these markers.

Of the 56 cores, 28 were positive and 28 were negative for ER, 19 werepositive and 37 were negative for PR, and there were 14 HER2 positiveand 42 HER2 negative cores. Tumors with high expression of HER2 were lowor negative for ER and PR. Conversely, ER and PR staining were high inbreast cancer specimens with low or absent HER2. Whereas increasingtumor grade was associated with an increase in positivity for OPN-cstaining according to mean combined score, mean percent positive, andmean intensity (compare FIGS. 3B and 3C), ER and PR were substantiallyreduced in higher grade tumors. HER2 was slightly higher in grades 2 and3 than in grade 1 (FIG. 4A).

To determine whether the cytoplasmic OPN-c staining of breast carcinomaswas correlated with the membrane staining of HER2 or the nuclearstaining of ER and PR, the mean combined scores were compared accordingto Spearman's rank correlation coefficient (SRCC) [16]. There was nosignificant correlation between OPN-c and any of these three breastmarkers. Only a tentative correlation according to SRCC was found withHER2 (FIG. 4B). As a metastasis gene, OPN-c is a marker for differentcharacteristics of breast cancer than these growth factor receptors.

It was then determined whether OPN-c has diagnostic value in conjunctionwith ER, PR and HER2. Applying a logistic regression modeling approach[17, 18], OPN-c was the best single predictor of a grade 3 cancer (ROCscore 0.809; ROC score for ER 0.575, for PR 0.570, for HER2 0.551).Furthermore, the combination of OPN-c, ER and HER2 can diagnose grade2-3 tumors with high reliability (ROC score 0.979) (FIG. 4C). In thismodel, high OPN-c and HER2 scores correspond to a higher likelihood ofgrade 2-3, and higher ER corresponds to a lower probability of grade2-3. PR had poor predictive value.

Triple-negative breast cancer (missing ER, PR, and HER2) lacks thebenefit of a specific therapy and is associated with a difficult riskassessment [19]. For all grades of breast cancer, 14% of the tissuecores that were positive for OPN-c were not stained by ER, PR, or HER2.Selectively for the highest grade of cancer (grade 3), approximately 35%of the cases that were stained by anti-OPN-c were negative for all ofthe 3 other breast cancer markers tested (Table 3). Hence, OPN-c detectsa larger fraction of breast cancers than do ER, PR or HER2. It cantherefore indicate transformation in cases where the other three markersfail.

TABLE 3 Histochemical staining for OPN-c, ER, PR, and HER2. OPNc + 3OPNc +, 1 OPNc +, 2 OPNc +, 3 OPNc −, 1 OPNc −, 2 OPNc −, 3 tumorreceptors receptor receptors receptors all receptor receptors receptorsgrade n negative positive positive positive negative positive positivepositive 1 16   0% 12.5% 43.8%   0% 6.3% 6.3% 25.0%  6.3% 2 23  8.7%56.5% 8.7%   0% 13.0%  13.0%    0%   0% 3 17 35.3% 41.2% 23.5%   0%   0%  0%   0%   0% all 56 14.3% 39.3% 23.2%   0% 7.1% 7.1% 7.1% 1.8% gradesThe comparison of percentages of samples staining positively forosteopontin-c versus all three growth factor receptors in breastcarcinoma tissue array cores indicates that OPN-c is a more sensitivemarker. It also detects a fraction of triple negative breast cancers.Note that samples with one or two of the receptors staining positivelyhave been omitted for clarity.

Example 5 OPN-c Expression in Other Cancers

Beside breast cancer, osteopontin has been associated with theprogression of other malignancies, including colon cancer, ovariancancer, hepatocellular carcinoma, and glioblastoma. A limited number ofcolon tumors (n=6), their surrounding tissues (n=4), and colitisspecimens (n=3) were obtained and expression levels of osteopontinsplice variants analyzed using real-time RT-PCR.

While the sample numbers are still small, they indicate that aproportion of colorectal carcinomas produce spliced osteopontin (Table4). In fact, while OPN-a was very low in the tumors (m=0.024, range0-0.088) and moderate in the adjacent tissues (m=0.258, range 0-0.543),the RNA message for OPN-c was expressed at very high levels in thetumors (m=0.474, range 0-1.5) and at moderate levels in the adjacenttissues (m=0.255, range 0-1.020). In the colitis samples, the expressionof OPN-a was low, while OPN-b and OPN-c were barely detectable.

In contrast to breast tissues, where normal specimens are accessiblefrom reduction mammoplasties, this type of source is not available forcolon. The comparison to colitis samples is somewhat compromised becauseseveral cell types of the immune system can produce osteopontin,although physiologic splicing of the osteopontin message has not yetbeen described. These results indicate that OPN-c is a marker ofinvasiveness not only for breast cancers, but also for othermalignancies. Therefore, the methods and kits provided herein can beused not only for breast cancers, but other cancers as well (e.g., thosethat express OPN such as adrenocortical cancer, ameloblastoma, ampullarycancer, bladder cancer, bone cancer, breast cancer, cervical cancer,cholangioma, colorectal cancer, endometrial cancer, esophageal cancer,gastric cancer, glioma, granular call tumor, head and neck cancer,hepatocellular cancer, hydatiform mole, lung cancer, lymphoma, melanoma,mesothelioma, myeloma, neuroblastoma, oral cancer, osteochondroma,osteosarcoma, ovarian cancer, pancreatic cancer, pilomatricoma, prostatecancer, renal cell cancer, salivary gland tumor, soft tissue tumors,Spitz naevus, squamous cell cancer, teratoid cancer, and thyroidcancer). OPN-c can be evaluated in such tissues using the methodsprovided herein.

TABLE 4 Osteopontin expression in other samples. OPN-a OPN-b OPN-cadjacent adjacent adjacent Diagnosis tumor normal tumor normal tumornormal colorectal tumors adenocarcinoma n = 6 n = 4 n = 6 n = 4 n = 6 n= 4    0-0.088 0-0.543 0-0.034 0-0.069 0-1.5 0-1.020 colitis m = 0.024 m= 0.258 m = 0.009 m = 0.018 m = 0.474 m = 0.255 diverticulitis n = 1 nan = 1 na n = 1 na m = 0.039 m = 0 m = 0 ulcerative colitis n = 3 na n =3 na n = 3 na 0.059-0.166 0-0.006 0-0.045 m = 0.106 m = 0.002 m = 0.015

Example 6 Analysis of Subjects with Known Clinical Outcome

Clinical samples from subjects with breast cancer (such as DCIS) whohave a known clinical outcome (for example, but not limited to, alive ordead after 1, 3, or 5 years; relapsed or relapse-free after 1, 3, or 5years; suffering metastases or metastasis-free after a given time frame)are analyzed for the presence of OPN-c as described in Example 1. Theskilled artisan will recognize that clinical outcomes can be defined inmultiple ways.

The clinical samples will be staged and graded as described inExample 1. The presence of OPN-c will be detected using antibodies asdescribed in Example 1 (e.g., staining scored using a typical 1+ to 3+scale or a 0 to 300 scale). In some examples, the clinical samples arealso analyzed for ER, PR, and HER2 expression as described in Example 1.The ability of OPN-c staining of the breast cancer samples to predictthe outcome of the patient will be determined. It is expected thatsubjects having greater OPN-c expression (e.g., 3 on a 1+ to 3+ scale)will have a greater likelihood of adverse outcome (e.g., dead after 1,3, or 5 years of diagnosis), as compared to subjects having less or noOPN-c expression (e.g., 0 or 1 on a 1+ to 3+ scale). Subjects havingless OPN-c expression (e.g., 1 on a 1+ to 3+ scale) will have a greaterlikelihood of a favorable outcome (e.g., alive after 1, 3, or 5 years ofdiagnosis).

Example 7 Grading of Breast Cancer in Humans

This example describes particular methods that can be used to gradebreast cancer, such as DCIS, in a human subject. However, one skilled inthe art will appreciate that similar methods can be used, and that othercancer samples (e.g., from the colon or other tissue listed in Example5) can be used in place of breast cancer samples. In some examples, suchgrading is performed before treating the subject.

Breast cancer cell or tissue samples are obtained from the subject. Forexample, a sample can be obtained from a tissue biopsy or needleaspirate using routine methods. In some examples, if a tissue biopsysample is used, 1-100 μg of tissue can be obtained, for example using afine needle aspirate. In particular examples, samples are used directly,or can be manipulated prior to use, for example, by fixing (e.g., usingformalin) or embedding (e.g., in plastic or paraffin). In some examples,RNA or proteins are isolated from the tissue using routine methods (forexample using a commercial kit).

In one example, OPN-c protein levels are determined in a breast cancersample obtained from the subject. The breast cancer sample, such as atissue or cell sample present on a substrate (such as a microscopeslide) is incubated with an OPN-c antibody in an appropriate buffer fora time sufficient for the antibody to bind to OPN-c in the sample. TheOPN-c/antibody complexes are detected, for example using microscopy. Insome examples, the breast cancer sample (or other sample from the samesubject) is analyzed for ER, PR, or HER2 protein levels usingappropriate antibodies.

The antibody/protein complexes can be detected by a label on theantibody, or with a secondary labeled antibody. The resulting stainedarray slides can be scored by light microscopy by a pathologistaccording to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+  Strong reactivity: Dark brown to black staining isusually, but not always, of tumor cell membranes in a complete membranepattern, producing a thick outline of the cell. above background levelCytoplasmic reactivity may be absent or may be moderately intense whenwhether it is complete or membrane staining is very intense.Submembranous cytoplasmic incomplete circumferential accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0   No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The resulting score can be used to grade, stage, or prognose the tumor(e.g., see Table 1).

In other examples, the relative amount of OPN-c (or ER, PR,HER2)/antibody complexes in the breast cancer sample from the subjectcan be compared to a reference value, such as a relative amount of OPN-c(or ER, PR, HER2)/antibody complexes present in a normal breast sampleor a breast cancer sample from a subject having a known grade of tumor(e.g., grade 1, 2 or 3). The presence of significantly greaterOPN-c/antibody complexes in the test breast cancer sample relative tothe control indicates that the subject has a higher grade or stagecancer (e.g., grade 3) and a worse prognosis, while the presence of lessOPN-c/antibody complexes indicates that the subject has a lower grade orstage cancer (e.g., grade 1) and a better prognosis.

In one example, OPN-c mRNA expression levels are determined in a breastcancer sample obtained from the subject. cDNA is generated from the RNAisolated from the breast cancer sample (for example using a commercialreverse transcription kit). OPN-c cDNA is amplified using appropriateprimers (for example using primers having a detectable label), and theresulting OPN-c amplicons detected. The relative amount of OPN-camplicons in the breast cancer sample can be normalized (e.g., to ahousekeeping gene) and compared to a reference value, such as a relativeamount of OPN-c amplicons present in a normal breast sample or a breastcancer sample from a subject having a known grade or stage of tumor(e.g., grade 1, 2 or 3). The presence of higher amounts of OPN-camplicons in the test breast cancer sample indicates that the subjecthas a higher grade or stage cancer (e.g., grade 3) and a worseprognosis, while the presence of less OPN-c amplicons indicates that thesubject has a lower grade or stage cancer (e.g., grade 1) and a betterprognosis.

REFERENCES

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In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only examples of the invention and shouldnot be taken as limiting the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A kit consisting of: (a) a nucleic acid probe or primerthat specifically hybridizes to an estrogen receptor (ER) nucleic acidmolecule; a nucleic acid probe or primer that specifically hybridizes toa progesterone receptor (PR) nucleic acid molecule; a nucleic acid probeor primer that specifically hybridizes to a human epidermal growthfactor receptor 2 (HER2) nucleic acid molecule; and a nucleic acid probeor primer that specifically hybridizes to an OPN-c nucleic acidmolecule; and/or (b) an antibody that specifically binds estrogenreceptor (ER); an antibody that specifically binds progesterone receptor(PR); an antibody that specifically binds HER2; and an antibody thatspecifically binds OPN-c.
 2. The kit of claim 1, wherein the nucleicacid probe or primer that specifically hybridizes to an OPN-c nucleicacid molecule consists of the nucleic acid sequences of SEQ ID NO: 5and/or SEQ ID NO:
 6. 3. The kit of claim 1, wherein the nucleic acidprobe or primer that specifically hybridizes to an estrogen receptor(ER) nucleic acid molecule specifically hybridizes under high stringencyconditions to the nucleic acid set forth in GenBank Accession No.BC128574.1 or NM_007956.4, wherein high stringency conditions comprisehybridization at about 42° C. in 5×SSC and washes performed at about 65°C.
 4. The kit of claim 1, wherein the nucleic acid probe or primer thatspecifically hybridizes to a progesterone receptor (PR) nucleic acidmolecule specifically hybridizes under high stringency conditions to thenucleic acid set forth in GenBank Accession No. AF016381.1 or M68915.1,wherein high stringency conditions comprise hybridization at about 42°C. in 5×SSC and washes performed at about 65° C.
 5. The kit of claim 1,wherein the nucleic acid probe or primer that specifically hybridizes toa HER2 nucleic acid molecule specifically hybridizes under highstringency conditions to the nucleic acid set forth in GenBank AccessionNo. NM_001005862.1 or NM_004448.2, wherein high stringency conditionscomprise hybridization at about 42° C. in 5×SSC and washes performed atabout 65° C.
 6. A kit consisting of: (a) a nucleic acid probe or primerthat specifically hybridizes to an estrogen receptor (ER) nucleic acidmolecule; a nucleic acid probe or primer that specifically hybridizes toa progesterone receptor (PR) nucleic acid molecule; a nucleic acid probeor primer that specifically hybridizes to a human epidermal growthfactor receptor 2 (HER2) nucleic acid molecule; and a nucleic acid probeor primer that specifically hybridizes to an OPN-c nucleic acidmolecule; and/or (b) an antibody that specifically binds estrogenreceptor (ER); an antibody that specifically binds progesterone receptor(PR); an antibody that specifically binds HER2; and an antibody thatspecifically binds OPN-c, wherein (a) and/or (b) is optionally combinedwith (i) one or more control samples with known amounts of ER, PR, HER2,and OPN-c, and/or (ii) a nucleic acid probe or primer that specificallyhybridizes to a housekeeping gene or a message of a housekeeping gene;and/or an antibody that specifically binds to a protein expressed by ahousekeeping gene.
 7. The kit of claim 6, wherein the nucleic acid probeor primer that specifically hybridizes to an OPN-c nucleic acid moleculeconsists of the nucleic acid sequences of SEQ ID NO: 5 and/or SEQ ID NO:6.
 8. The kit of claim 6, wherein the nucleic acid probe or primer thatspecifically hybridizes to an estrogen receptor (ER) nucleic acidmolecule specifically hybridizes under high stringency conditions to thenucleic acid set forth in GenBank Accession No. BC128574.1 orNM_007956.4, wherein high stringency conditions comprise hybridizationat about 42° C. in 5×SSC and washes performed at about 65° C.
 9. The kitof claim 6, wherein the nucleic acid probe or primer that specificallyhybridizes to a progesterone receptor (PR) nucleic acid moleculespecifically hybridizes under high stringency conditions to the nucleicacid set forth in GenBank Accession No. AF016381.1 or M68915.1, whereinhigh stringency conditions comprise hybridization at about 42° C. in5×SSC and washes performed at about 65° C.
 10. The kit of claim 6,wherein the nucleic acid probe or primer that specifically hybridizes toa HER2 nucleic acid molecule specifically hybridizes under highstringency conditions to the nucleic acid set forth in GenBank AccessionNo. NM_001005862.1 or NM_004448.2, wherein high stringency conditionscomprise hybridization at about 42° C. in 5×SSC and washes performed atabout 65° C.